Affinity chromatography has long been used to identify the protein targets of small-molecule drugs and other biomolecules. While an essential tool for biochemical research, affinity chromatography can often be labor intensive and time consuming. Recently the yeast three-hybrid assay, a derivative of the two-hybrid assay, was introduced as a straightforward, in vivo alternative to affinity chromatography (1,2). The yeast two-hybrid system relies on the interaction of two fusion proteins to bring about the transcriptional activation of a reporter gene thus identifying protein-protein interactions in an in vivo system (2). The subsequently developed yeast three-hybrid system screens for a small molecule-protein interaction based on the principle that small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine (3). In the three-hybrid assay, protein-small-molecule interactions are detected as reconstitution of a transcriptional activator (“TA”) and subsequent transcription of a reporter gene (4–7). A dimeric small-molecule ligand bridges the DNA-binding domain (“DBD”) of the TA, which is fused to the receptor for one ligand, and the activation domain (“AD”) of the TA, which is fused to the receptor for the other ligand. For affinity chromatography applications, one ligand-receptor pair is used as an anchor and the other is the small-molecule-protein interaction being investigated. While the yeast three-hybrid assay is quite powerful, a bacterial equivalent would increase the number of proteins that could be tested by several orders of magnitude because the transformation efficiency and doubling time of E. coli is significantly greater than that of S. cerevisiae. In addition, there may be applications where it is advantageous to test a eukaryotic protein in a prokaryotic environment where many pathways are not conserved.
However, the yeast three-hybrid assay cannot be transferred directly to bacteria. The components of the transcription machinery and the mechanism of transcriptional activation differ significantly between bacteria and yeast. Ligand-receptor pairs often are organism specific because of cell permeability, toxicity, or other interactions with the cellular milieu. Bacterial two-hybrid assays have only begun to be developed in the past few years (8) and to date only initial efforts toward the design of a robust bacterial three-hybrid system have been reported (9, 10). Described below is the first robust small-molecule bacterial three-hybrid system.